Many studies on anti-bacterial/antiviral surfaces have been conducted to prevent epidemic spread worldwide. Several nanoparticles such as those composed of silver and copper are known to have antiviral properties. In this study, we developed copper oxide (CuO) nanoparticle-incorporated nanofibers to inactivate or remove viruses. The CuO nanoparticle-incorporated nanofiber was fabricated with a hydrophobic polymer-polyvinylpyrrolidone (PVP)-using electrospinning, and CuO nanoparticles were exposed from the PVP polymer surface by etching the nanofiber with oxygen plasma. The fabrication conditions of electrospinning and oxygen plasma etching were investigated by scanning electron microscopy (SEM), and field emission transmission electron microscopy (FETEM)/ energy dispersive spectrometry (EDS). H1N1 virus was utilized as the target sample and quantified by RT-qPCR. The antiviral efficacy of CuO nanoparticle-incorporated nanofibers was compared against bare CuO nanoparticles. Overall, 70% of the viruses were inactivated after CuO nanoparticle-incorporated nanofibers were incubated with 10² pfu/mL of H1N1 virus solution for 4 h. This indicates that the developed CuO nanoparticle-incorporated nanofibers have noticeable antiviral efficacy. As the developed CuO nanoparticle-incorporated nanofibers exerted promising antiviral effects against H1N1 virus, it is expected to benefit global health by preventing epidemic spread.